So, You Think An Electron Microscope Is There To Make Things Look Bigger?

“Well, of course”- I can hear someone saying. “It can make things look as much as 200, 000 times bigger”. Problem is, the first sentence is wrong, and the second one is meaningless!

On quick and thoughtless answers to simple questions

I had just been admitted as an MSc student at UCL, and was attending my first tutorial with the course tutor, Professor (now Emeritus) Giorgio Gabella. For those who do not know him, he is, among other things, one of the most amazing electron microscopists around. I was very technically inclined, had been using an electron microscope without assistance since I was 20, so I felt pretty confident that there were not many things to learn on the subject. Ah, the folly of youth! At some point, Prof. Gabella asked, “what is it the advantage of using an electron microscope, instead of a light microscope?” I wasted no time, and immediately replied “higher magnification!”- only to receive the answer “No!”. What? “You can blow up any photo to any size you like. You can choose any magnification you want when printing a photo- you don’t need an expensive electron microscope for that!”

Resolution

So what was the answer? “Higher resolution”, of course! Remember Abbe’s law from 1873? It is expressed in the form of a simple and not very scary formula: d=?/2NA, where d is the resolution limit, ? is the wavelength of the light used, and NA is the numerical aperture of the lens. This law defines resolution as a function of the wavelength used, and the smallest possible detail that can be seen using light is 200 nm. So this is the resolution of the light microscope. Today there are ways to circumvent the limitations of Abbe’s law while still using light (superresolution microscopy) but that’s another story (and another series of articles) altogether.

What’s so great about this scope?

So what’s so great about the electron microscope? It uses electrons instead of photons, and as they have shorter wavelengths, a much increased resolution is achieved, down to 0.2 nm. Of course its ‘lenses’ are not real optical lenses, they are coils in which the electromagnetic fields can focus electron beams, and sample preparation is totally different to anything you have experienced using any form of light microscope. Suffice to say, it’s not for the faint-hearted!  And there are drawbacks associated with it, such as potential artifacts. It’s the exact opposite of live cell imaging- the samples are not just dead and fixed, they are hard-as-stone and heavy-metal impregnated.

New tricks for an old dog

The electron microscope was invented shortly before WWII, became very popular in the ‘70s and ‘80s, and seemed to have given all it had to give about 20 years ago. However, the huge increase in computer storage and processing capacity of image data in the last few years has made it fashionable again, as people can now make really really, really long sequences of ultrathin serial sections, and then combine them and analyze them in 3-D, pretty much like the software of a confocal microscope does.

You have been warned

Of course, unlike the confocal, the ultrathin sections are real, physical sections, which have to be cut and collected with the help of an ultramicrotome. You have been warned.  Correlative Light and Electron Microscopy- ie doing what you want to do at the light level and then at the electron level- is another application that is in vogue in the last several years.

Bar none

So, is it totally pointless to say that we get ‘higher magnification’ with an electron microscope? We can salvage this, if we use the word ‘useable’. A higher useable magnification equals a higher resolution.

And what about ‘200,000 x’? That is usually pointless. Sometimes people in lectures use slides of photos which have come from books.  In the book it says “200,000x magnification”- and in the lecture they repeat the same. Well, it can’t be. In the book the photo is 10×10 cm and on the wall it’s 100×100 cm. The same is true in electronic media. The actual magnification depends on the size of your monitor. So it’s better not to talk about ‘magnification’. Use a scale bar. You can’t go wrong. The scale bar is 20 ?m on your monitor, a piece of paper, or the wall, and this is why scale bars are the only way you should use to indicate, well, scale, in all your image applications. Bar none.